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N. Pozdeyev, S.S. Chaurasia, R. Haque, Y. Du, H. Fu, P.M. Iuvone; Melatonin Synthesis in Chicken Retina: Regulation of Arylalkylamine N–Acetyltransferase Binding to 14–3–3 Proteins by Light and Darkness . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3992.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Arylalkylamine–N–acetyltransferase (AANAT) is a key regulatory enzyme that drives the circadian rhythm of melatonin synthesis. Light exposure at night causes rapid decrease in AANAT activity and melatonin synthesis in the retina and pineal gland so that melatonin functions strictly as a chemical signal for darkness. We studied the molecular mechanism of AANAT regulation by light in the chicken retina. Methods: The effect of light exposure was studied in retinas of two week old chickens injected intravitreally with lactacystin, an inhibitor of proteasomal proteolysis, to prevent AANAT degradation. Soluble retinal proteins were separated by gel filtration chromatography and specific AANAT activity was measured in fractions as well as in protein extracts. Formation of AANAT/14–3–3 complexes was studied by co–immunoprecipitation of AANAT with 14–3–3 antibodies. Results: Light exposure significantly increased the apparent Km of AANAT for its substrate, tryptamine. During separation of retinal soluble proteins by gel filtration chromatography, AANAT activity eluted as three peaks with different apparent molecular weights. A low molecular weight fraction (∼20kDa) corresponded to the free, monomeric enzyme. A second peak of activity was identified as an AANAT/14–3–3 complex (molecular weight ∼70–90 kDa). The Km of the AANAT/14–3–3 complex for tryptamine was 0.02 mM, which is approximately 15 times lower than the Km of the free enzyme. The high molecular weight peak (>150 kDa) of AANAT has a Km similar to that of the monomeric enzyme; its physical nature is unknown, but it may represent enzyme that is ubiquitinated or docked to the proteasome. In dark adapted retinas, most of AANAT exists as a complex with 14–3–3 proteins. Unexpected light at night caused dissociation of AANAT/14–3–3 complex, accompanied by an increase in monomeric and high molecular weight forms of enzyme. An incubation of protein extract with catalytic subunit of protein kinase A partially reversed this effect of light. Conclusions: Our results are consistent with the hypothesis that AANAT is phosphorylated in the dark, favoring the binding to 14–3–3. This interaction protects the enzyme from degradation and increases its affinity for substrate. Upon light exposure, the enzyme dissociates from 14–3–3, is inactivated and dephosphorylated, and is targeted for degradation by the proteasome. The high Km of the monomeric enzyme suggests that it has little activity in situ with physiological concentrations of substrate, insuring that melatonin synthesis is rapidly terminated upon light exposure.
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